1. Field of the Invention
The present invention relates to the plasmid pTR2030 and to derivatives thereof. More specifically, the present invention relates to the plasmid pTR2030 which carries genetic determinants for phage resistance to group N streptococci and to derivatives of this plasmid which also carry the same genetic determinants.
2. Description of the Prior Art
Production of cheese and cultured dairy products has long relied on the fermentation of milk by group N streptococci. Members of this group, composed of Streptococcus lactis, S. cremoris, and S. lactis subsp. diacetylactis, are directly responsible for the acid development, flavor production, and often coagulum characteristics in mesophilic dairy fermentations. Because efficient milk fermentations are dependent on the growth and activity of the lactic streptococci, great care is exercised to prepare starter cultures that are highly active and uncontaminated with undesirable microorganisms or bacteriophages. However, the fermentation process itself is nonaseptic, occurring in open vats with a nonsterile medium, pasteurized milk. It is therefore highly susceptible to contamination with bacteriophages. For the majority of strains of lactic streptococci employed in commercial dairy fermentations, lytic bacteriophages capable of halting growth and acid production can appear within 1 to 2 days after introducing the culture into the cheese plant. Although bacteriophage contamination of numerous industrial fermentations has been observed, the destructive role of bacteriophages in milk fermentations is without parallel in other fermentation processes.
Historically, milk fermentations relied on starter cultures composed of undefined mixtures of lactic streptococci propagated without knowledge of, or protection from, bacteriophages. Natural phage contamination in these cultures established an equilibrium of evolving bacteriophages and phage-resistant variants. These cultures were highly variable in day-to-day levels of acid production, but remained moderately active and could be used continuously in small fermentation factories. Over the past 20 years, starter culture failures due to bacteriophage infection have become prevalent throughout the dairy industry. Increasing demand for cultured milk products in recent years has necessitated increases in both production capacity and process efficiency such that larger volumes of milk are processed, cheese vats are filled repeatedly within a single day, and total processing time is shortened. This modernization of the industry concurrently increased the probability of phage contamination and further dictated the use of defined mixtures of lactic streptococci capable of uniform and rapid rates of acid production. With the selection of highly fermentative lactic streptococci and their propagation under aseptic conditions (in the absence of bacteriophages), the majority of cultures now used by the industry have become highly susceptible to bacteriophage attack upon introduction into the cheese factory.
To cope with bacteriophage problems a number of successful methods have been developed to minimize phage action during commercial milk fermentations. Through the use of concentrated cultures, aseptic bulk starter vessels and phage-inhibitory media (see for example, U.S. Pat. No. 4,282,255), the starter culture can be protected from bacteriophage infection prior to vat inoculation. However, phage contamination cannot be prevented following entrance into the fermentation vat. Therefore, emphasis for protection of the culture shifts to minimizing prolific phage-host interactions through rotation of phage-unrelated strains or use of phage-resistant mutants in multiple-strain starters. Although, in theory, strain rotation should minimize developing phage populations within the plant, in practice it has proved difficult to identify strains that demonstrate completely different patterns of phage sensitivity. Estimates of the total number of different, phage-unrelated lactic streptococci approximate 25 strains worldwide. Considering the small number of phage-unrelated strains available, the choice of strains for incorporation into rotation programs is severely limited. Similarly, few phage-unrelated strains are available for construction of multiple-strian starters containing composites of 4 to 6 strains.
A decade ago, Sandine, W. E. et al., J. Milk Food Technol. 35, 176 (1972) emphasized the need to isolate new strains of lactic streptococci for use in the dairy industry. Foremost among the criteria for selection of these strains was resistance to existing bacteriophages. It is now recognized that some strains of lactic streptococci are not attacked by any phage when challenged with large collections of laboratory phage banks, or when used on a continuous, long-term basis in commercial fermentations. These reports demonstrate the existence of lactic streptococci that are not sensitive to bacteriophage attack, in spite of devastating phage pressure such as that which routinely occurs within the factory environment. However, to date, only a limited number of phage-insensitive strains have been identified and studied for mechanisms of phage resistance.
Several mechanisms of phage resistance in group N streptococci have been identified and appear to be plasmid-associated. McKay, L. L. et al., Appl. Environ. Microbiol. 47, 68 (1984) describe a 40 megadalton plasmid, pNP40, found in S. lactis subsp. diacetylactis DRC3. When the plasmid was conjugally transferred to S. lactis C2, transconjugants were isolated which were resistant to phage c2 at 21.degree. C. and 32.degree. C., but not at 37.degree. C. It was found that the resistance to c2 was not due to an inhibition of phage adsorption or to a classical modification-restriction, but was suggested to be a temperature-sensitive DNase. The authors concluded that the genetic determinant for this resistance was located on pNP40.
Sanders, M.E., et al., Appl. Environ. Microbiol. 47, 979 (1984) reported that S. lactis ME2 was insensitive to a variety of phages. The authors determined that this insensitivity was the result of several temperature-sensitive mechanisms including: (a) prevention of phage adsorption, (b) the modification-restriction system, and (c) suppression of phage development. The authors reported that the genetic determinants for some of the mechanisms may be found on plasmids, but that some appeared to be found on the chromosomes.
Sanders, M. E., et al., Appl. Environ. Microbiol. 46, 1125 (1983) disclose that S. lactis ME2 contains a plasmid, pME0030, which codes for a function that prevents phage adsorption. Sanders, M. E. et al., Appl. Environ. Microbiol. 42, 944 (1981) disclose that a 10 megadalton plasmid found in S. cremoris KH codes for a modification-restriction system which enables this strain to be resistant to phage c2.
Gonzalez, C. F. et al., Appl. Environ. Microbiol. 49, 627 (1985) reported that two transconjugants of two matings of S. lactis SLA 2.24 or SLA 3.15 and S. lactis subsp. diacetylactis SLA 3.10 or SLA 3.23, respectively, showed temperature-independent phage resistance which was not due to adsorption or restriction in phage growth. Physical evidence for plasmid involvement was not obtained.
The identification or creation of plasmids encoding phage resistance in group N streptococci is necessary in order to genetically engineer strains that meet industrial criteria for fermentative capabilities and long-term phage resistance. The present invention provides for a plasmid which confers phage resistance to group N streptococci. Group N streptococci containing the plasmid or a derivative thereof are useful for formulating starter cultures which can be used for the production of cheese and cultured dairy products.